System and apparatus for sequential processing of analytes

ABSTRACT

An apparatus and system are provided for simultaneously analyzing a plurality of analytes anchored to microparticles. Microparticles each having a uniform population of a single kind of analyte attached are disposed as a substantially immobilized planar array inside of a flow chamber where steps of an analytical process are carried out by delivering a sequence of processing reagents to the microparticles by a fluidic system under microprocessor control. In response to such process steps, an optical signal is generated at the surface of each microparticle which is characteristic of the interaction between the analyte carried by the microparticle and the delivered processing reagent. The plurality of analytes are simultaneously analyzed by collecting and recording images of the optical signals generated by all the microparticles in the planar array. A key feature of the invention is the correlation of the sequence of optical signals generated by each microparticle in the planar array during the analytical process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/693,658, filed on Dec. 4, 2012 which is a continuation of U.S.application Ser. No. 11/974,239, filed on Oct. 12, 2007, now U.S. Pat.No. 8,361,713, which is a division of U.S. application Ser. No.11/207,443 filed on Aug. 18, 2005, now U.S. Pat. No. 7,282,370, which isa division of U.S. application Ser. No. 09/908,131 filed on Jul. 17,2001, now U.S. Pat. No. 6,969,488, which is a division of U.S.application Ser. No. 09/424,028 filed on Nov. 16, 1999, now U.S. Pat.No. 6,406,848, which is a 371 of PCT Application No. PCT/US98/11224filed on May 22, 1998, all of which are incorporated herein by referencein their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledILLINC153C3.TXT created Apr. 4, 2014, which is approximately 5 KB insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to systems and apparatus for carryingout large scale parallel reactions on solid phase supports, and moreparticularly, to systems and apparatus for monitoring and carrying outreactions on arrays of microparticles.

BACKGROUND

The desire to understand and analyze complex chemical and biologicalsystems has led to the development of analytical techniques that employparallelization and miniaturization of analyte processing, e.g. Graberet al., Current Opinion in Biotechnology, 9:14-18 (1998); Fodor et al.,Nature, 364:555-556 (1993); Meier-Ewert et al., Nature, 361:375-376(1993); Taylor et al., Nucleic Acids Research, 25:3164-3168 (1997);Garner et al., BioTechniques, 14:112-115 (1993); Lam et al., Nature,354:82-84 (1991); Ohlmeyer et al., Proc. Natl. Acad. Sci.,90:10922-10926 (1993); DeRisi et al., Science, 278:680-686 (1997);Wodicka et al., Nature Biotechnology, 15:1359-1367 (1997); and the like.

Many of these techniques employ microparticles for synthesizing analytesor for capturing analytes for subsequent analysis, e.g. Lam et al.(cited above); Benkovic et al., International patent applicationPCT/US95/03355; Gavin et al., International patent applicationPCT/EP97/02039; Brenner et al., International patent applicationPCT/US96/09513, and the like. Even though the properties of differenttypes of microparticles can vary widely, microparticles generallyfacilitate the construction and manipulation of large repertoires ofanalytes with minimal reagent and/or sample consumption. However,handling and manipulating large numbers of microparticles, e.g. tens tohundreds of thousands, for carrying out specific chemical and/orbiochemical analyses gives rise to many difficulties, including whethersufficient signal is generated on individual microparticles fordetection, how to track individual microparticles through multiple stepsof a process, mechanical strength of microparticles under pressure orflow conditions, the ability to uniformly deliver reagents tomicroparticles for carrying out steps of an analytical process, whetherclumping or other inappropriate interaction of microparticles and/orreagents occurs, whether or adsorption of analytes and/or processingreagents onto vessel walls occurs, whether protein reagents or analytesdenature causing a disruption of reagent distribution and access,whether adjacent microparticles will interact, e.g. to degrade orobscure a signal or to inhibit reagent access, and the like.

In view of these difficulties, it would be desirable to provide a systemand apparatus for handling and processing multiple solid phase supports,such as populations of microparticles. It would be especially desirableif such system and apparatus permitted the tracking and analysis ofmultiple analytes anchored to separate microparticles through a sequenceof several processing and/or analysis steps.

SUMMARY OF THE INVENTION

Accordingly, objects of our invention include, but are not limited to,providing a system and apparatus for sequentially delivering reagents toa population of analytes anchored to a separate microparticles;providing an apparatus for simultaneously monitoring the interactions ofprocessing reagents and analytes on the surfaces of microparticlesdisposed in a planar array; providing an apparatus for detecting opticalsignals generated by, or as the result of, interactions of processingreagents and analytes on the surfaces of microparticles disposed in aplanar array; providing an apparatus for detecting pluralities ofoptical signals, each such plurality being generated at the surface ofthe same microparticle as a result of interactions between processingreagents and an analyte anchored to the surface of such microparticle;providing an apparatus for simultaneously tracking the positions ofindividual microparticles in a population of microparticles disposed ina flow chamber as a closely packed planar array; and providing a systemand apparatus for simultaneously analyzing the nucleotide sequences of apopulation of polynucleotides anchored to microparticles disposed in aplanar array in a flow chamber.

Our invention achieves these and other objects with an apparatuscomprising a flow chamber for disposing a population of microparticlesin a planar array; fluidic means for sequentially delivering processingreagents from one or more reagent reservoirs to the flow chamber; anddetection means for detecting a sequence of optical signals from each ofthe microparticles of the population. Preferably, the sequences ofoptical signals are generated as a result of a multi-step analyticalprocess, such as nucleic acid sequence analysis.

In one aspect, the invention provides a system for simultaneouslymonitoring a population of analytes which includes the apparatus of theinvention, microparticles carrying the analytes, and software means forprocessing images of, and/or optical signals generated by, themicroparticles when disposed in a planar array. Preferably, the flowchamber includes constraining means for restricting the movement ofmicroparticles during cycles of reagent delivery.

In another aspect, the invention includes a system for simultaneouslyanalyzing the nucleotide sequences of a population of polynucleotides.Copies of each kind of polynucleotide in the population are sorted ontoand anchored to one or more microparticles so that a population ofloaded microparticles is formed. Loaded microparticles are disposed in aplanar array in a flow chamber through which processing reagents aresequentially delivered to the loaded microparticles from one or morereagent reservoirs by a fluidic means. Optical signals generated by, orproduced as a result of, the interaction of processing reagents andpolynucleotides on the microparticles are imaged by a detection means.Preferably, when analysis includes determining the nucleotide sequenceof a portion of each polynucleotide on the different microparticles,massively parallel signature sequencing (MPSS) analysis is employed,e.g. as described in Albrecht et al, International patent applicationPCT/US97/09472.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a schematic representation of a flow chamber and fluidicsand detection systems for observing a planar array of microparticlesloaded with analyte molecules, such as cDNA molecules for sequencing.

FIG. 1 b is a schematic of a preferred holder for a flow chamber.

FIG. 2 a is bilateral cut away view of a flow chamber.

FIG. 2 b is a top view of a flow chamber.

FIG. 2 c is an illustration of microparticles being loaded into a flowchamber.

FIGS. 3 a, 3 b, 3 c, and 3 d schematically illustrate microparticleconstraining means for a flow chamber.

FIG. 4 is a schematic representation of a device for loadingmicroparticles into a flow chamber.

FIG. 5 is a schematic representation of a fluidics system for use withthe invention.

FIGS. 6A and 6B schematically illustrate top-lighting and back-lightingapproaches for determining microparticle centers in an array.

FIG. 7 schematically illustrates the assignment of pixels tomicroparticles for data processing.

FIG. 8 is a flow chart summarizing operation of the system of theinvention.

DEFINITIONS

“Complement” or “tag complement” as used herein in reference tooligonucleotide tags refers to an oligonucleotide to which aoligonucleotide tag specifically hybridizes to form a perfectly matchedduplex or triplex. In embodiments where specific hybridization resultsin a triplex, the oligonucleotide tag may be selected to be eitherdouble stranded or single stranded. Thus, where triplexes are formed,the term “complement” is meant to encompass either a double strandedcomplement of a single stranded oligonucleotide tag or a single strandedcomplement of a double stranded oligonucleotide tag.

The term “oligonucleotide” as used herein includes linear oligomers ofnatural or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptidenucleic acids (PNAs), and the like, capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomerto-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like.Usually monomers are linked by phospho diester bonds or analogs thereofto form oligonucleotides ranging in size from a few monomeric units,e.g. 3-4, to several tens of monomeric units, e.g. 40-60. Whenever anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′□3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine,unless otherwise noted. Usually oligonucleotides of the inventioncomprise the four natural nucleotides; however, they may also comprisenon-natural nucleotide analogs. It is clear to those skilled in the artwhen oligonucleotides having natural or non-natural nucleotides may beemployed, e.g. where processing by enzymes is called for, usuallyoligonucleotides consisting of natural nucleotides are required.

“Perfectly matched” in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one other such that every nucleotide in each strandundergoes Watson-Crick basepairing with a nucleotide in the otherstrand. The term also comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, and thelike, that may be employed. In reference to a triplex, the term meansthat the triplex consists of a perfectly matched duplex and a thirdstrand in which every nucleotide undergoes Hoogsteen or reverseHoogsteen association with a basepair of the perfectly matched duplex.Conversely, a “mismatch” in a duplex between a tag and anoligonucleotide means that a pair or triplet of nucleotides in theduplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/orreverse Hoogsteen bonding.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2^(nd) Ed. (Freeman, San Francisco, 1992). “Analogs” inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90:543-584 (1990), or the like, with the only provisothat they are capable of specific hybridization. Such analogs includesynthetic nucleosides designed to enhance binding properties, reducecomplexity, increase specificity, and the like.

As used herein “sequence determination” or “determining a nucleotidesequence” in reference to polynucleotides includes determination ofpartial as well as full sequence information of the polynucleotide. Thatis, the term includes sequence comparisons, fingerprinting, and likelevels of information about a target polynucleotide, as well as theexpress identification and ordering of nucleosides, usually eachnucleoside, in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. For example, in some embodiments sequence determinationmay be effected by identifying the ordering and locations of a singletype, of nucleotide, e.g. cytosines, within the target polynucleotide“CATCGC . . . ” so that its sequence is represented as a binary code,e.g. “100101 . . . ” for “C-(not C)-(not C)-C-(not C)-C . . . ” and thelike.

As used herein, the term “complexity” in reference to a population ofpolynucleotides means the number of different species of moleculepresent in the population.

DETAILED DESCRIPTION OF THE INVENTION

The system and apparatus of the invention is particularly applicable tothe analysis of molecules that can be anchored in populations ofduplicate copies to particulate solid phase supports. That is, inaccordance with the invention, each analyte of a population is presenton at least one microparticle in a quantity sufficient for the type ofanalysis being performed. For example, if combinatorially synthesizedpeptides on the microparticles are screened against a soluble receptorprotein for detecting those that form stable complexes, the number ofpeptides available for binding on the surface of the microparticles mustbe large enough to generate a detectable signal when a binding eventoccurs. Of course, many additional factors well known in the art willpresent additional design constraints, such as the nature of the systemfor generating optical signals, the concentration of receptors, pH, saltconcentration, the density and accessibility of the peptides on themicroparticle surface, the solvent system employed, and the like.Analyte populations particularly relevant for use with the presentapparatus include combinatorial libraries synthesized on microparticlesupports, e.g as disclosed in Lam et al., Chem. Rev., 97:411-448 (1997);or Dower et al., U.S. Pat. No. 5,708,153, and polynucleotide librariessorted onto microparticle supports, e.g. as disclosed in Brenner (citedabove).

FIG. 1A is a schematic representation of an embodiment of the inventionfor detecting fluorescent signals. Flow chamber (100) having inlet(102), outlet (104) and planar cavity (106) holds microparticles in aplanar array from which optical signals (108) generated by analytesand/or reactants on microparticles can be collected and imaged. Flowchamber (100) is operationally associated with fluidic system (112) anddetection system (114), so that delivery of fluids and collection ofsignals is under control of computer (116). Preferably, optical signalsare collected by microscope (118) and are imaged onto a solid stateimaging device, such as charge-coupled device (CCD) (120) which iscapable of generating a digital image of the physical image of themicroparticle array with sufficient resolution for individualmicroparticles to be distinguished. For fluorescent signals, detectionsystem (114) usually includes appropriate bandpass filter (122) foroptical signal (108), bandpass filter (124) for excitation beam (128)generated by light source (126), and other standard components. Asillustrated, a conventional fluorescence microscope is preferred whichis configured for epiillumination. There is a great deal of guidance inthe art for selecting appropriate fluorescence microscopes, e.g Wang andTaylor, editors, Fluroescence Microscopy of Living Cells in Culture,Parts A and B, Methods in Cell Biology, Vols. 29 and 30 (Academic Press,New York, 1989).

A key feature of the invention is flow chamber (100). Body (130) of flowchamber (100) preferably comprised inlet (102), outlet (104) and planarcavity (106) which are formed by standard micromachining techniques,e.g. Ekstrom et al., International patent application PCT/SE91/00327;Brown, U.S. Pat. No. 4,911,782; Harrison et al, Anal. Chem. 64:1926-1932(1992); and the like. Transparent plate (132) is sealingly attached tobody (130) to form an operational flow chamber (100). Body (130) may beconstructed from several different materials including glass, silicon,polyethylene, polyester, teflon, other plastics, and the like.Preferably, transparent plate (132) is glass or quartz; and, when body(130) and transparent plate (132) are glass or silicon, transparentplate (132) is preferably attached to body (130) by anodic bonding, e.g.Pomerantz, U.S. Pat. No. 3,397,279. Key functions of the flow chamberinclude i) holding a population of microparticles in a substantiallyimmobilized planar array, or monolayer, during a sequence of processingsteps, ii) ensuring that processing reagents can access eachmicroparticle during each step of a process, and iii) minimizingprocessing reagent usage. The degree of immobilization required may varyamong different embodiments. Generally, more movement of microparticleswithin a planar array increases the computational and measurement burdenof tracking positions of microparticles by image processing software.Design trade-offs therefore exist between the use of image processingsoftware and the use of physical and/or chemical means for constrainingmicroparticle movement. Preferably, physical and/or chemical means areemployed to constrain microparticle movement within the planar array ofmicro particles in flow chamber (100). Such means are referred to hereinas “movement constraining means.” Most preferably, physical, ormechanical, movement constraining means are employed.

Preferably, microparticles are disposed in flow chamber (100) in aclosely packed planar array. As used herein, “closely packed” inreference to a planar array means either that the number ofmicroparticles per unit area of a planar array is at least eightypercent of the number of micro particles in a hexagonal array of equalarea, or that the average distance between centers of adjacentmicroparticles is less than two microparticle diameters. As used herein,a “hexagonal” array of microparticles means a planar array ofmicroparticles in which every microparticle in the array contacts atleast six other adjacent microparticles, as shown in FIG. 3A.

Additional features of flow chamber (100) of a preferred embodiment areillustrated in FIGS. 2A through 2C. FIG. 2A is a cross sectional viewalong a longitudinal plane that bisects flow chamber (100). The sameview, in a more abstracted rendition, is shown in FIG. 2C. In bothFigures, inlet (102) fluidly communicates with planar cavity (106) andoutlet (104). Microparticles (200) carrying analytes enter inlet (102)and are carried by a suspending buffer to planar cavity (106) where theybecome packed against dam (202) which prevents the microparticles fromexiting the flow chamber through outlet (104). Structurally, dam (202)may be formed by a sudden reduction of the vertical dimension of planarcavity (106). Preferably, vertical dimension (204) of planar cavity(106) is selected so that microparticles (200) are constrained to aplane, i.e. a monolayer, when they pack against dam (202). Morepreferably, vertical dimension (204) is selected to be between about 120to 150 percent of the diameter of the microparticles employed. Forexample, when microparticles are employed that have diameters of 5 μm,vertical dimension (204) may be 7 μm. Magnetic microparticles may beconstrained to a plane and constrained from movement by applying amagnetic field so that the microparticles are attracted to the ceilingor to the floor of planar cavity (106). Width (206) of planar cavity(106) is not a critical dimension; however, for convenience andefficiency, width (206) may be selected to correspond to the dimensionsof the signal collection region of detection system (114). Such regionslabeled 1 through k in FIG. 2B are referred to herein as “tiles.” Thatis, the region of planar cavity (106) occupied by microparticles may bedivided into non-overlapping areas, referred to as “tiles,” that coverthe entire occupied region. FIG. 2B, which is a top view of the flowchamber of FIG. 2A, also shows inlet (102), planar cavity (106), dam(202), and outlet (104) that lie in sequence along axis (217) of flowchamber (100).

Many movement constraining means may be selected for use with the flowchamber, either alone or in combination. Such means include loadingmicroparticles with trace amounts of a chemically reactive species whichmay be activated and cross-linked; providing physical, or mechanicalstructures, such as ridges, within the flow chamber; providingmagnetically responsive microparticles which may be immobilized by anexternal magnetic field; providing a second population of microparticlesthat are loaded into a flow chamber after the analyte-containingpopulation, which forces the analyte containing population against dam(202); and the like. Exemplary chemically reactive species for use withnucleic acid analytes are disclosed in Summerton et al., U.S. Pat. No.4,123,610; Gamper et al., J. Mol. Biol., 197:349-362 (1987); Hearst,Ann. Rev. Phys. Chem. 39:291-315 (1988); Pieles et al., Nucleic AcidsResearch, 17:8967-8978 (1989); and the like.

Preferably, microparticle movement is constrained by providing a flowchamber with planar cavity (106) containing a plurality of ridgesrunning parallel to axis (217) of the flow chamber, i.e. parallel to thedirection of reagent flow, so that microparticles are arranged intorows, which may be single-file, or several microparticles wide, as shownin FIGS. 3A, 3B, 3C, and 3D. The particular selection may depend onseveral factors, including the degree of immobilization desired,constraints imposed by the fabrication technique used to construct theflow chamber, the amount of reagent access desired, the degree to whichflow resistance or back-pressure can be tolerated, and the like. FIGS.3A and 3B illustrate two possible distances between parallel ridges. InFIG. 3A, the distance is selected to permit maximal packing of microparticles into a hexagonal array, and in FIG. 3B, the distance isselected for less efficient packing, but for increased reagent access tomicroparticle surfaces.

In some embodiments, such as those employing enzymatic processes, theinner surfaces of flow chamber (100) may be passivated, that is, treatedto render such surfaces inert and/or non-adsorbing with respect toenzymes. The type of treatment depends on the sensitivity of the enzymesused in the process, and their affinity for the surfaces. Surfacetreatments include silanization, e.g. with commercially availablereagents (Pierce, Rockford, Ill.); and/or adsorption of various blockingpolymers, such as poly-a-alanine, polyglycine, polyadenylic acid,polymaleimide, polyvinylpyrrolidone, or the like, e.g. Shoffner et al.,Nucleic Acids Research, 24:375-379 (1996). Preferably, glass innersurfaces of flow chamber (100) are covalently coated with a neutralcoating, such as allyl methacrylate, using the technique disclosed inSandoval et al., U.S. Pat. No. 5,326,738, which is incorporated byreference.

FIG. 1B illustrates flow chamber (100) mounted between holders (140) and(142) which sealingly connect inlet (102) to inlet tubing (144) andoutlet (104) to outlet tubing (146), respectively. Preferably, holder(140) contains a rotary valve (not shown) operated by actuator (148)that shunts fluid flowing through inlet tubing (144) to inlet (102) orto waste line (150). Such a valve minimizes the amount of processreagent from a previous step that must be passed through flow chamber(100) prior to the initiation of the next process step. That is, such arotary valve permits reagent in inlet tubing (144) to be shunted towaste and replaced by processing reagent required for the next step inthe process being executed. Preferably, for use in DNA analysis, peltierblock (152) is employed to control temperature in flow chamber (100) andthe entire assembly including flow chamber (100) and peltier block (152)is mounted on xyz-stage (154) which is under control of computer (116).

Preferably, microparticles are loaded into flow chamber (100) prior toattachment of holders (140) and (142) and the initiation of processingsteps. FIG. 4 illustrates a microparticle loader for loadingmicroparticles into flow chamber (100). Flow chamber (100) is mountedbetween holders (400), (402), (404), and (406). Holders (400) and (402)sealingly clamp onto the inlet end (101) of flow chamber (100) andholders (404) and (406) sealingly clamp onto the outlet end (103) offlow chamber (100) so that inlet tubing (408) is in fluid communicationwith outlet tubing (410) when the microparticle loader is assembled.Inlet tubing (408) is connected to syringe (416) which is used to drivefluid through flow chamber (100). Holder (400) is constructed to haveconical passage (412) which narrows to match the diameter of inlet (102)of flow chamber (100). After assembly of holders (400), (402), (404),and (406) a suspension of micro particles is placed in the conicalpassage after which fitting (414) is sealingly connected to holder(400). Fluid pressure and flow generated by syringe (416) then drivesthe microparticles into planar cavity (106) and against dam (202). In apreferred embodiment which employs 5 μm diameter GMA microparticlescarrying DNA, approximately 500 thousand microparticles are loaded intoflow chamber (100) by placing 5 μL of a 100 thousand microparticle/μLsolution (TE buffer, pH 8.0, Sambrook et al., Molecular Cloning, SecondEdition (Cold Spring Harbor Laboratory, New York, 1989)) in conicalpassage (412), attaching fitting (414), and using syringe (416) to drivethe microparticles through inlet (102) and into planar cavity (106).After loading, holders (400), (402), (404), and (406) are removed fromflow chamber (100), which is then mounted on the apparatus as shown inFIG. 1B.

Preferably, process reagents are delivered to flow chamber (100) by thefluidic system illustrated in FIG. 5 which has the capacity to handlemany different reagents for complex analytical processes. In theillustrated embodiment, which is used in connection with DNA sequencing,the fluidics system may accommodate up to 38 reagents, including washbuffers, rinses, enzymes, hybridization probes, adaptors, and the like.Preferably, the function of the fluidics system is the sequentialmetering of selected processing reagents to flow chamber (100). Inlet(102) of flow chamber (100) is sealingly connected to holder (140) whichcontains rotary valve (actuator shown as 148) (not shown in FIG. 5). Thefunction of the rotary valve is described above. A variety of means maybe employed for moving processing reagents from reservoirs, throughtubing, and into flow chamber (100), including gravity feed, pressurefeed, and pumps, e.g. peristaltic, syringe, and the like. Preferably,common syringe pump (500) is employed for removing predetermined amountsprocessing reagents from reservoirs and for forcing such reagentsthrough flow chamber (100) at a predetermined flow rate. Under controlof computer (116), pump (500) in operational association with valveblock (502) and rotary valve (504) removes a predetermined amount ofprocessing reagent from a selected reservoir by siphoning reagent out ofthe reservoir on the out-stroke of plunger (501) of pump (500). On theinstroke of plunger (501), rotary valve (504) directs processing reagentfrom tubing (503) to reservoir (505) of pump (500). On the out-stroke ofplunger (501), state of rotary valve (504) is changed to directprocessing reagent from reservoir (505) to inlet tubing (144). Tubing(503) connects rotary valve (504) with manifold (508) which, in turn, isconnected to a plurality (five shown) of banks of zero dead volumevalves (506). Zero dead volume valves (506) connect individualreservoirs holding processing reagents to a common passageway (not shownin FIG. 5) that runs through each of the banks of valves connecting tomanifold (508).

A preferred zero dead volume valve is described in U.S. Pat. Nos.4,558,845 and 4,703,913, which are incorporated by reference. Processreagents from reservoirs (514) are distributed to the banks of deadvolume valves by way of manifold (510). Alternative valve blocks forcontrolling delivery of process reagents to flow chamber (100) includethe valve matrix disclosed in U.S. Pat. No. 5,203,368.

An important feature of detection means (114) of the invention is theability to keep track of individual microparticles through multipleprocess steps and/or cycles. In connection with such tracking, detectionmeans (114) periodically records optical characteristics of individualmicroparticles that provide a close approximation microparticle centers.Preferably, when trans-illumination, or “back lighting” of flow chamber(100) is possible, the optical characteristic is the focused back lightfrom the microparticles. That is, in reference to FIG. 6A, back light(600) passes vertically through flow chamber (100) where it is focusedby microparticles (602) onto focal plane (604). The image of focal plane(604) in this configuration appears as a field of bright points, whereeach point is located at the approximate center of its correspondingmicroparticle. In an epiillumination system, light from above flowchamber (100), i.e. “top light (610),” is directed from a verticaldirection onto microparticles (602) where it scatters from the topsurface of the microparticles. In this configuration, the opticalcharacteristic is the scatter center of a microparticle. Thus, an imageis collected from the plane containing scatter centers (612) resultingfrom such top lighting. As with focused back lighting, the image of thescatter centers provides a convenient way to readily determine theapproximate centers of the microparticles.

In the preferred image processing approach, once microparticle centers(700) are determined, pixels (702) are assigned for determiningcharacteristics, e.g. intensity, of an optical signal generated at eachmicroparticle (602). The size of micro particle (602) and pixel areadetermine how many pixels are assigned to each microparticle. In makingsuch an assignment, important factors include the degree to which thecalculated center of a microparticle (as described above) is likely todeviate from the geometric center, the extent to which optical signalcollected from the edge of an image contains spurious information (e.g.signal from an overlapping or adjacent microparticle), the uniformity ofmicroparticle diameter and shape, and the like. In the preferredapparatus of the invention, 5 μm diameter microparticles are employedand the pixel dimensions of the CCD detector are about 0.9 μm×0.9 μm.Thus, nine pixels fit easily within the interior of a microparticleimage with a margin of at least about 1 μm between any pixel and theedge of the microparticle image. In the preferred embodiment, an initialpixel is assigned which encloses the computed center of a microparticle,e.g. pixel “5” in FIG. 7. Thereafter, additional pixels are assigned,usually the immediately adjacent pixels. Preferably, the value of theoptical signal generated by a process at the surface of a microparticleis the average value of the optical signals collected by pixels assignedto that microparticle.

The general operation of the system of the preferred embodiment issummarized by the flow chart of FIG. 8. At the start (800) of ananalysis, microparticles with anchored analytes have been loaded intoflow chamber (100) which has been operationally mounted in holders 140and 142. The initial operation is the calibration of the microparticlefocal plane (802). That is, the vertical, or “z”, position of thexyz-stage is determined which optimizes the focus of either the scattercenters of the microparticles, i.e. the microparticle tops fortop-lighting, or the focus points of the microparticles forback-lighting. The optimization is carried out by a conventionalautofocusing algorithm which provides an image contrast functionconstructed from a predetermined sample of regions within a collectedimage. For example, the contrast function may be evaluated iterativelyfor sequence of z-positions so that the differences of successive valuesof the contrast function can be determined. These are tested until adifference is found below a predetermined threshold, which is taken asthe maximum of the contrast function. Focal plane location is taken asthe z position which maximizes the image contrast function. Suchcalibration is carried out for each tile, if more than one tile isemployed, so that a correction table is constructed of changes in stagesetting values with respect to the settings of the first tile that arerequired to bring the system into focus upon translation to subsequenttiles. These values are stored by computer (116).

After calibration, process steps are initiated (804) by way of afluidics controller operationally associated with computer (116). Afterprocess steps (804) are completed, stage settings are adjusted to placethe first tile into focus using the autofocus algorithm (806), whichplaces the focal plane of the microscope objective approximately at thetops of the microparticles. Stage settings are then adjusted (808) tobring the focal plane of the microscope objective to the approximatecenters of the microparticles, as illustrated (606) in FIGS. 6A and 6B.The amount of stage movement in this re-focusing depends on the diameterof the microparticles being used. After appropriate selection of filters(124) and (122), a fluorescent image of the first tile is collected(810) and transferred to data server (812). Fluorescent images arecollected on the plane of the microparticle centers because ofimperfections in the planar array. That is, microparticles in planarcavity (106) do not lie in a perfect planar array for a variety ofreasons. For example, some microparticles are elevated above others as aresult of packing into the flow chamber; there is some variability inthe size and shape of the microparticles; and, the floor of planarcavity (106) may be uneven. After the fluorescent image is collected,the focal plane of the microscope objective is returned (814) to themicroparticle focal plane, where another image is collected (816) forthe purpose of computing microparticle centers as described above. Theimage of micro particle centers is transferred to data server (812)where data processor (818) assigns pixels of the fluorescent image toeach microparticle center, as described above. After the image of microparticle centers is collected (816), the stage is moved so that an imageof the next tile can be collected (822). If there are no further tilesof microparticles (820), then the next steps and/or cycles of theprocess are executed (826). If there are no further process steps (824),then the process is complete and the apparatus is placed in a holdingmode.

Optical signals collected in the course of analysis may be generated bya variety of mechanisms, including absorption and fluorescence,chemiluminescence, electrochemiluminescence, or bioluminescenceemission. Extensive guidance is available for selecting appropriateoptical signaling means, e.g. Kessler, editor, Nonradioactive Labelingand Detection of Biomolecules (Springer-Verlag, Berlin); Keller andManak, DNA Probes, Second Edition (Stockton Press, New York, 1993); andthe like. Preferably, optical signals generated in processing steps arefluorescence emissions.

Microparticles

An important feature of the system of the invention is the use of microparticles for carrying analytes. A variety of microparticles may beemployed depending on particular applications. Generally, microparticlesmust consist of a material compatible with the reagents and chemistry ofthe process steps being carried out and microparticle must besubstantially mechanically rigid so that they retain their shape andsize during process steps. Preferably, as used herein, the term“substantially mechanically rigid” means that microparticles neitherswell nor contract by more than ten percent (as measure by diameter) inany process solvent or reagent. Preferably, microparticles are microspheres of uniform size, i.e. microparticles are monodisperse. Morepreferably, the diameters of spherical microparticles have a coefficientof variation less than five percent, and most preferably, less than twopercent. Microparticle diameters are in the range of from 0.1 μm to 100μm. Preferably, microparticle diameters range from 1 μm to 20 μm. Mostpreferably, microparticle diameters are in the range of 1 to 5 μm.Suitable microparticle materials include inorganic support materialssuch as glass, e.g. controlled-pore glass, Balltoni beads; silica,zirconia, and the like, e.g. Weetall, Methods in Enzymology, 44:134-148(1976); and organic support materials such as highly cross-linkedpolystyrene, polyacrylate, polymethylmethacrylate, glycidylmethacrylate(GMA), Dynabeads (Dynal, Oslo, Norway), and the like, Rembaum et al.,U.S. Pat. No. 4,046,720; Hodge and Sherrington, editors, pages 435-456,Polymer-supported Reactions in Organic Synthesis (Wiley & Sons, NewYork, 1980); Andrus et al., U.S. Pat. No. 5,047,524; and the like.

Attaching Identical Copies of Polynucleotides to Microparticles by SolidPhase Cloning

In a preferred embodiment of the invention, identical copies ofpolynucleotides from a population are anchored to separatemicroparticles by solid phase cloning, i.e. the use of oligonucleotidetags for sorting polynucleotides onto microparticles such that only thesame kind of polynucleotide will be attached to the same microparticle,e.g. Brenner, U.S. Pat. No. 5,604,097, which is incorporated byreference. This condition is accomplished by taking a sample of the fullensemble of tag-polynucleotide conjugates. (It is acceptable thatidentical polynucleotides have different tags, as it merely results inthe same polynucleotide being operated on or analyzed twice in twodifferent locations.) Such sampling can be carried out eitherovertly—for example, by taking a small volume from a largermixture—after the tags have been attached to the polynucleotides, it canbe carried out inherently as a secondary effect of the techniques usedto process the polynucleotides and tags, or sampling can be carried outboth overtly and as an inherent part of processing steps.

Oligonucleotide tags for use with the invention are members of aminimally cross-hybridizing set of oligonucleotides. The sequences ofoligonucleotides of such a set differ from the sequences of every othermember of the same set by at least two nucleotides: Thus, each member ofsuch a set cannot form a duplex (or triplex) with the complement of anyother member with less than two mismatches. Complements ofoligonucleotide tags of the invention, referred to herein as “tagcomplements,” may comprise natural nucleotides or non-natural nucleotideanalogs. Tag complements are attached to microparticles.

Minimally cross-hybridizing sets of oligonucleotide tags and tagcomplements may be synthesized either combinatorially or individuallydepending on the size of the set desired and the degree to whichcross-hybridization is sought to be minimized (or stated another way,the degree to which specificity is sought to be enhanced). For example,a minimally cross-hybridizing set may consist of a set of individuallysynthesized 10-mer sequences that differ from each other by at least 4nucleotides, such set having a maximum size of 332 (when composed of 3kinds of nucleotides and counted using a computer program such asdisclosed in Appendix Ic of International patent applicationPCT/US96/09513). Alternatively, a minimally cross-hybridizing set ofoligonucleotide tags may also be assembled combinatorially from subunitswhich themselves are selected from a minimally cross-hybridizing set.For example, a set of minimally cross-hybridizing 12-mers differing fromone another by at least three nucleotides may be synthesized byassembling 3 subunits selected from a set of minimally cross-hybridizing4-mers that each differ from one another by three 3 nucleotides. Such anembodiment gives a maximally sized set of 9³, or 729, 12-mers, “9” isnumber of oligonucleotides generated by the computer program of AppendixIa of International patent application PCT/US96/09513, which assumes, aswith the 10-mers, that only 3 of the 4 different types of nucleotidesare used. The set is described as “maximal” because the computerprograms disclosed in International patent application PCT/US96/09513provide the largest set for a given input (e.g. length, composition,difference in number of nucleotides between members). Additionalminimally crosshybridizing sets may be formed from subsets of suchcalculated sets.

When synthesized combinatorially, an oligonucleotide tag of theinvention preferably consists of a plurality of subunits, each subunitconsisting of an oligonucleotide of 3 to 9 nucleotides in length whereineach subunit is selected from the same minimally cross-hybridizing set.In such embodiments, the number of oligonucleotide tags availabledepends on the number of subunits per tag and on the length of thesubunits.

As used herein in reference to oligonucleotide tags and tag complements,the term “repertoire” means the set of minimally cross-hybridizing setof oligonucleotides that make up the tags in a particular embodiment orthe corresponding set of tag complements.

Preferably, in constructing a cDNA library where substantially alldifferent cDNAs have different tags, a tag repertoire is employed whosecomplexity, or number of distinct tags, greatly exceeds the total numberof mRNAs extracted from a cell or tissue sample. Preferably, thecomplexity of the tag repertoire is at least 10 times that of thepolynucleotide population; and more preferably, the complexity of thetag repertoire is at least 100 times that of the polynucleotidepopulation. Below, a protocol is disclosed for cDNA library constructionusing a primer mixture that contains a full repertoire of exemplary9-word tags. Such a mixture of tag-containing primers has a complexityof 8⁹, or about 1.34×10⁸. As indicated by Winslow et al., Nucleic AcidsResearch, 19:3251-3253 (1991), mRNA for library construction can beextracted from as few as 5×10⁵ mammalian cells. Since a single mammaliancell contains about 5×10⁵ copies of mRNA molecules of about 3.4×10⁴different kinds, by standard techniques one can isolate the mRNA fromabout 100 cells, or (theoretically) about 5×10⁷ mRNA molecules.Comparing this number to the complexity of the primer mixture shows thatwithout any additional steps, and even assuming that mRNAs are convertedinto cDNAs with perfect efficiency (1% efficiency or less is moreaccurate), the cDNA library construction protocol results in apopulation containing no more than 37% of the total number of differenttags. That is, without any overt sampling step at all, the protocolinherently generates a sample that comprises 37%, or less, of the tagrepertoire. The probability of obtaining a double under these conditionsis about 5%, which is within the preferred range. With mRNA from 10cells, the fraction of the tag repertoire sampled is reduced to only3.7%, even assuming that all the processing steps take place at 100%efficiency. In fact, the efficiencies of the processing steps forconstructing cDNA libraries are very low, a “rule of thumb” being thatgood library should contain about 10⁸ cDNA clones from mRNA extractedfrom 10⁶ mammalian cells.

Use of larger amounts of mRNA in the above protocol, or for largeramounts of polynucleotides in general, where the number of suchmolecules exceeds the complexity of the tag repertoire, atag-polynucleotide conjugate mixture potentially contains every possiblepairing of tags and types of mRNA or polynucleotide. In such cases,overt sampling may be implemented by removing a sample volume after aserial dilution of the starting mixture of tag-polynucleotideconjugates. The amount of dilution required depends on the amount ofstarting material and the efficiencies of the processing steps, whichare readily estimated.

If mRNA were extracted from 10⁶ cells (which would correspond to about0.5 μg of poly(A)⁺ RNA), and if primers were present in about 10-100fold concentration excess—as is called for in a typical protocol, e.g.Sambrook et al, Molecular Cloning, Second Edition, page 8.61 [10 μL 1.8kb mRNA at 1 mg/mL equals about 1.68×10⁻¹¹ moles and 10 μL 18-mer primerat 1 mg/mL equals about 1.68×10⁻⁹ moles], then the total number oftag-polynucleotide conjugates in a cDNA library would simply be equal toor less than the starting number of mRNAs, or about 5×10¹¹ vectorscontaining tag-polynucleotide conjugates—again this assumes that eachstep in cDNA construction—first strand synthesis, second strandsynthesis, ligation into a vector—occurs with perfect efficiency, whichis a very conservative estimate. The actual number is significantlyless.

If a sample of n tag-polynucleotide conjugates are randomly drawn from areaction mixture—as could be effected by taking a sample volume, theprobability of drawing conjugates having the same tag is described bythe Poisson distribution, P(r)=e^(λ)(λ)^(r)/r, where r is the number ofconjugates having the same tag and λ=np, where p is the probability of agiven tag being selected. If n=10⁶ and p=1/(1.34×10⁸), then λ=0.00746and P(2)=2.76×10⁻⁵. Thus, a sample of one million molecules gives riseto an expected number of doubles well within the preferred range. Such asample is readily obtained as follows: Assume that the 5×10¹¹ mRNAs areperfectly converted into 5×10¹¹ vectors with tag-cDNA conjugates asinserts and that the 5×10¹¹ vectors are in a reaction solution having avolume of 100 μl. Four 10-fold serial dilutions may be carried out bytransferring 10 μl from the original solution into a vessel containing90 μl of an appropriate buffer, such as TE. This process may be repeatedfor three additional 5 dilutions to obtain a 100 μl solution containing5×10⁵ vector molecules per μl. A 2 μl aliquot from this solution yields10⁶ vectors containing tag-cDNA conjugates as inserts. This sample isthen amplified by straight forward transformation of a competent hostcell followed by culturing.

Of course, as mentioned above, no step in the above process proceedswith perfect efficiency. In particular, when vectors are employed toamplify a sample of tag-polynucleotide conjugates, the step oftransforming a host is very inefficient. Usually, no more than 1% of thevectors are taken up by the host and replicated. Thus, for such a methodof amplification, even fewer dilutions would be required to obtain asample of 10⁶ conjugates.

A repertoire of oligonucleotide tags can be conjugated to a populationof polynucleotides in a number of ways, including direct enzymaticligation, amplification, e.g. via PCR, using primers containing the tagsequences, and the like. The initial ligating step produces a very largepopulation of tag-polynucleotide conjugates such that a single tag isgenerally attached to many different polynucleotides. However, as notedabove, by taking a sufficiently small sample of the conjugates, theprobability of obtaining “doubles,” i.e. the same tag on two differentpolynucleotides, can be made negligible. Generally, the larger thesample the greater the probability of obtaining a double. Thus, a designtrade-off exists between selecting a large sample of tag-polynucleotideconjugates—which, for example, ensures adequate coverage of a targetpolynucleotide in a shotgun sequencing operation or adequaterepresentation of a rapidly changing mRNA pool, and selecting a smallsample which ensures that a minimal number of doubles will be present.In most embodiments, the presence of doubles merely adds an additionalsource of noise or, in the case of sequencing, a minor complication inscanning and signal processing, as microparticles giving multiplefluorescent signals can simply be ignored.

As used herein, the term “substantially all” in reference to attachingtags to molecules, especially polynucleotides, is meant to reflect thestatistical nature of the sampling procedure employed to obtain apopulation of tag-molecule conjugates essentially free of doubles. Themeaning of substantially all in terms of actual percentages oftag-molecule conjugates depends on how the tags are being employed.Preferably, for nucleic acid sequencing, substantially all means that atleast eighty percent of the polynucleotides have unique tags attached.More preferably, it means that at least ninety percent of thepolynucleotides have unique tags attached. Still more preferably, itmeans that at least ninety-five percent of the polynucleotides haveunique tags attached. And, most preferably, it means that at leastninety-nine percent of the polynucleotides have unique tags attached.

Tags can be conjugated to cDNAs of existing libraries by standardcloning methods. cDNAs are excised from their existing vector, isolated,and then ligated into a vector containing a repertoire of tags.Preferably, the tag-containing vector is linearized by cleaving with tworestriction enzymes so that the excised cDNAs can be ligated in apredetermined orientation. The concentration of the linearizedtag-containing vector is in substantial excess over that of the cDNAinserts so that ligation provides an inherent sampling of tags.

A general method for exposing the single stranded tag afteramplification involves digesting a target polynucleotide-containingconjugate with the 5′→3′ exonuclease activity of T4 DNA polymerase, or alike enzyme, e.g. as described in Kuijper et at., Gene, 112:147-155(1992). When used in the presence of a single deoxynucleosidetriphosphate, such a polymerase will cleave nucleotides from 3′ recessedends present on the non-template strand of a double stranded fragmentuntil a complement of the single deoxynucleoside triphosphate is reachedon the template strand. When such a nucleotide is reached the 5′→3′digestion effectively ceases, as the polymerase's extension activityadds nucleotides at a higher rate than the excision activity removesnucleotides. Consequently, single stranded tags constructed with threenucleotides are readily prepared for loading onto solid phase supports.

After the oligonucleotide tags are prepared for specific hybridization,e.g. by rendering them single stranded as described above, thepolynucleotides are mixed with microparticles containing thecomplementary sequences of the tags under conditions that favor theformation of perfectly matched duplexes between the tags and theircomplements. There is extensive guidance in the literature for creatingthese conditions. Exemplary references providing such guidance includeWetmur, Critical Reviews in Biochemistry and Molecular Biology,26:227-259 (1991); Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Edition (Cold Spring Harbor Laboratory, New York, 1989); andthe like. Preferably, the hybridization conditions are sufficientlystringent so that only perfectly matched sequences form stable duplexes.Under such conditions the polynucleotides specifically hybridizedthrough their tags may be ligated to the complementary sequencesattached to the microparticles. Finally, the microparticles are washedto remove polynucleotides with unligated and/or mismatched tags.

Preferably, for sequencing applications, standard CPG beads of diameterin the range of 20-50)μm are loaded with about 10⁵ polynucleotides, andglycidalmethacrylate (GMA) beads available from Bangs Laboratories(Carmel, Ind.) of diameter in the range of 5-10 μm are loaded with a fewtens of thousand polynucleotide, e.g. 4×10⁴ to 6×10⁴, to a hundredthousand polynucleotides.

DNA Sequencing

Polynucleotides loaded onto microparticles may be simultaneouslysequenced in the instant apparatus using a “base-by-base” DNA sequencingmethodology. Such sequencing methodology permits the stepwiseidentification of a sequence of nucleotides in a target polynucleotide,usually one base at a time, through successive cycles of treatment anddetection. Base-by-base approaches are disclosed in the followingreferences: Cheeseman, U.S. Pat. No. 5,302,509; Tsien et al.,International application WO 91/06678; Rosenthal et al., Internationalapplication WO 93/21340; Canard et al., Gene, 148:1-6 (1994); Metzker etal, Nucleic Acids Research, 22:4259-4267 (1994); and the like.Preferably, the base-by-base approach disclosed by Brenner in U.S. Pat.No. 5,599,675 is used with the apparatus of the invention to sequencepolynucleotides on a population of loaded microparticles disposed as aplanar array in the flow chamber. Accordingly, Brenner, U.S. Pat. No.5,599,675 is incorporated by reference. Preferably, the a population ofloaded microparticles for sequencing includes at least ten thousandloaded microparticles; more preferably, such a population includes atleast fifty thousand loaded microparticles; and still more preferably,such a population includes at least one hundred thousand loadedmicroparticles.

Preferably, the sequencing method of Brenner (cited above) is employedin the embodiment disclosed in Albrecht et al. International patentapplication PCT/US97/09472 which discloses the use of encoded adaptors.An encoded adaptor is a double stranded oligonucleotide comprising aprotruding strand and an oligonucleotide tag selected from a minimallycross-hybridizing set of oligonucleotides. Encoded adaptors whoseprotruding strands form perfectly matched duplexes with thecomplementary protruding strands of the target polynucleotide areligated. After ligation, the identity and ordering of the nucleotides inthe protruding strands are determined, or “decoded,” by specificallyhybridizing a labeled tag complement to its corresponding tag on theligated adaptor. Encoded adaptors may be used in an adaptor-based methodof DNA sequencing that includes repeated cycles of ligation,identification, and cleavage, such as the method described in Brenner(cited above). Briefly, such a method comprises the following steps: (a)ligating an encoded adaptor to an end of a polynucleotide, the encodedadaptor having a nuclease recognition site of a nuclease whose cleavagesite is separate from its recognition site; (b) identifying one or morenucleotides at the end of the polynucleotide by the identity of theencoded adaptor ligated thereto; (c) cleaving the polynucleotide with anuclease recognizing the nuclease recognition site of the encodedadaptor such that the polynucleotide is shortened by one or morenucleotides; and (d) repeating said steps (a) through (c) until saidnucleotide sequence of the polynucleotide is determined. In theidentification step, successive sets of tag complements are specificallyhybridized to the respective tags carried by encoded adaptors ligated tothe ends of the target polynucleotides, as described above. The type andsequence of nucleotides in the protruding strands of the polynucleotidesare identified by the label carried by the specifically hybridized tagcomplement and the set from which the tag complement came.

Construction and Sorting of cDNA Library for Signature Sequencing withEncoded Adaptors

In this example, a cDNA library is constructed in which anoligonucleotide tag consisting of 8 four-nucleotide “words” is attachedto each cDNA. As described above, the repertoire of oligonucleotide tagsof this size is sufficiently large (about 10⁸) so that if the cDNAs aresynthesized from a population of about 10⁶ mRNAs, then there is a highprobability that each cDNA will have a unique tag for sorting. AftermRNA extraction, first strand synthesis is carried out in the presenceof 5-Me-dCTP (to block certain cDNA restriction sites) and abiotinylated primer mixture containing the oligonucleotide tags. Afterconventional second strand synthesis, the tag-cDNA conjugates arecleaved with Dpn II (which is unaffected by the 5-Me-deoxycytosines),the biotinylated portions are separated from the reaction mixture usingstreptavidin-coated magnetic beads, and the tag cDNA conjugates arerecovered by cleaving them from the magnetic beads via a Bsm BI sitecarried by the biotinylated primer. The Bsm BI-Dpn II fragmentcontaining the tag cDNA conjugate is then inserted into a plasmid andamplified. After isolation of the plasmids, tag-cDNA conjugates areamplified out of the plasmids by PCR in the presence of 5-Me-dCTP, usingbiotinylated and fluorescently labeled primers containing predefinedrestriction endonuclease sites. After affinity purification withstreptavidin coated magnetic beads, the tag-cDNA conjugates are cleavedfrom the beads, treated with T4 DNA polymerase in the presence of dGTPto render the tags single stranded, and then combined with a repertoireof GMA beads having tag complements attached. After stringenthybridization and ligation, the GMA beads are sorted via FACS to producean enriched population of GMA beads loaded with cDNAs. The enrichedpopulation of loaded GMA beads are immobilized in a planar array in aflow chamber where base-by-base sequence takes place using encodedadaptors, as disclosed in Albrecht et al., International patentapplication PCT/US97/09472.

Approximately 5 μg of poly(A⁺) mRNA is extracted from DBY746 yeast cellsusing conventional protocols. First and second strand cDNA synthesis iscarried out by combining 100-150 pmoles of the following primer (SEQ IDNO: 1):

5′-biotin-ACTAATCGTCTCACTATTTAATTAA[W, W, W, G]₈ CC(T)₁₈V-3′with the poly(A+) mRNA using a Stratagene (La Jolla, Calif.) cDNASynthesis Kit in accordance with the manufacturer's protocol. Thisresults in cDNAs whose first stand deoxycytosines are methylated at the5-carbon position. In the above formula, “V” is G, C, or A, “[W,W,W,G]”is a four-nucleotide word selected from Table II of Brenner,International patent application PCT/US96/09513, the single underlinedportion is a Bsm BI recognition site, and the double underlined portionis a Pac I recognition site. After size fractionation (GOBCO-BRL cDNASize Fractionation Kit) using conventional protocols, the cDNAs aredigested with Dpn II (New England Bioscience, Beverly, Mass.) usingmanufacturer's protocol and affinity purified with streptavidin-coatedmagnetic beads (M-280 beads, Dynal A.S., Oslo, Norway). The DNA capturedby the beads is digested with Bsm BI to release the tag-cDNA conjugatesfor cloning into a modified pBCSK⁻ vector (Stratagene, La Jolla, Calif.)using standard protocols. The pBCSK⁻ vector is modified by adding a BbsI site by inserting the following fragment (SEQ ID NO: 2) into the KpnI/Eco RV digested vector.

CGAAGACCC 3′-CATGGCTTCTGGGGATA-5′Bsm BI/Dpn II digested tag-cDNA conjugate is inserted in the pBCSK⁻which is previously digested with Bbs I and Bam HI. After ligation, thevector is transfected into the manufacturer's recommended host foramplification.

After isolating the above pBCSK⁻ vector from a standard plasmidminiprep, the tag-cDNA conjugates are amplified by PCR in the presenceof S-Me-dCTP using 20-mer primers complementary to vector sequencesflanking the tag-cDNA insert. The “upstream” primer, i.e. adjacent tothe tag, is biotinylated and the “downstream” primer, i.e. adjacent tothe cDNA, is labeled with fluorescein. After amplification, the PCRproduct is affinity purified then cleaved with Pac I to releasefluorescently labeled tagcDNA conjugates. The tags of the conjugates arerendered single stranded by treating them with T4 DNA polymerase in thepresence of dGTP. After the reaction is quenched, the tag-cDNA conjugateis purified by phenol-chloroform extraction and combined with 5.5 mm GMAbeads carrying tag complements, each tag complement having a 5′phosphate. Hybridization is conducted under stringent conditions in thepresence of a thermal stable ligase so that only tags forming perfectlymatched duplexes with their complements are ligated. The GMA beads arewashed and the loaded beads are concentrated by FACS sorting, using thefluorescently labeled cDNAs to identify loaded GMA beads. The tag-cDNAconjugates attached to the GMA beads are digested with Dpn II to removethe fluorescent label and treated with alkaline phosphatase to preparethe cDNAs for sequencing. That is, phosphatase is used to remove the 5′phosphate from the ends of the cDNAs to prevent unwanted cDNA-cDNAligations by way of the palindromic Dpn II site.

The following cleavage adaptor (SEQ ID NO: 3) is ligated to the DpnII-digested and phosphatase treated cDNAs:

5′-pGATCAGCTGCTGCAAATTT pTCGACGACGTTTAAAAfter ligation, the 3′ phosphate is removed by alkaline phosphatase, the5′ strand of the cDNA is treated with T4 DNA kinase, and the nickbetween the cleavage adaptor and cDNA is ligated. After cleavage by BbvI, encoded adaptors are ligated to the ends of the cDNAs and the beadsare ready for loading into the flow chamber.

Ligation of the adaptors to the target polynucleotide is carried out ina mixture consisting of 5 μl beads (20 mg), 3 μL NEB 10× ligase buffer,5 μL adaptor mix (25 nM), 2.5 μL NEB T4 DNA ligase (2000 units/μL), and14.5 μL distilled water. The mixture is incubated at 16° C. for 30minutes, after which the beads are washed 3 times in TE (pH 8.0).

After centrifugation and removal of TE, the 3′ phosphates of the ligatedadaptors are removed by treating the polynucleotide-bead mixture withcalf intestinal alkaline phosphatase (CIP) (New England Biolabs,Beverly, Mass.), using the manufacturer's protocol. After removal of the3′ phosphates, the CIP may be inactivated by proteolytic digestion, e.g.using Pronase™ (available form Boeringer Mannhiem, Indianapolis, Ind.),or an equivalent protease, with the manufacturer's protocol. Thepolynucleotide-bead mixture is then washed, treated with a mixture of T4polynucleotide kinase and T4 DNA ligase (New England Biolabs, Beverly,Mass.) to add a 5′ phosphate at the gap between the targetpolynucleotide and the adaptor, and to complete the ligation of theadaptors to the target polynucleotide. The bead-polynucleotide mixtureis then washed in TE, diluted to a concentration of approximately 100thousand beads per μL, and 5 μL of the resulting solution is loaded intoa flow chamber with the help of the holders of FIG. 4.

The top strands of the following 16 sets of 64 encoded adaptors (SEQ IDNO: 4 through SEQ ID NO: 19) are each separately synthesized on anautomated DNA synthesizer (model 392 Applied Biosystems, Foster City)using standard methods. The bottom strand, which is the same for alladaptors, is synthesized separately then hybridized to the respectivetop strands:

SEQ ID NO. Encoded Adaptor 4 5′-pANNNTACAGCTGCATCCCttggcgctgaggpATGCACGCGTAGGG-5′ 5 5′-pNANNTACAGCTGCATCCCtgggcctgtaagpATGCACGCGTAGGG-5′ 6 5′-pCNNNTACAGCTGCATCCCttgacgggtctcpATGCACGCGTAGGG-5′ 7 S′-pNCNNTACAGCTGCATCCCtgcccgcacagtpATGCACGCGTAGGG-5′ 8 5′~pGNNNTACAGCTGCATCCCttcgcctcggacpATGCACGCGTAGGG-5′ 9 5′-pNGNNTACAGCTGCATCCCtgatccgctagcpATGCACGCGTAGGG-S′ 10 5′-pTNNNTACAGCTGCATCCCttccgaacccgcpATGCACGCGTAGGG-5′ 11 5′-pNTNNTACAGCTGCATCCCtgagggggatagpATGCACGCGTAGGG-5′ 12 5′-pNNANTACAGCTGCATCCCttcccgctacacpATGCACGCGTAGGG-S′ 13 5′-pNNNATACAGCTGCATCCCtgactccccgagpATGCACGCGTAGGG-5′ 14 5′-pNNCNTACAGCTGCATCCCtgtgttgcgcggpATGCACGCGTAGGG-5′ 15 5′-pNNNCTACAGCTGCATCCCtctacagcagcgpATGCACGCGTAGGG-5′ 16 5′-pNNGNTACAGCTGCATCCCtgtcgcgtcgttpATGCACGCGTAGGG-5′ 17 5′-pNNNGTACAGCTGCATCCCtcggagcaacctpATGCACGCGTAGGG-5′ 18 5′-pNNTNTACAGCTGCATCCCtggtgaccgtagpATGCACGCGTAGGG-5′ 19 5′-pNNNTTACAGCTGCATCCCtcccctgtcggapATGCACGCGTAGGG-5′

where N and p are as defined above, and the nucleotides indicated inlower case letters are the 12-mer oligonucleotide tags. Each tag differsfrom every other by 6 nucleotides. Equal molar quantities of eachadaptor are combined in NEB #2 restriction buffer (New England Biolabs,Beverly, Mass.) to form a mixture at a concentration of 1000 pmol/μL.

Each of the 16 tag complements are separately synthesized asamino-derivatized oligonucleotides and are each labeled with afluorescein molecule (using an NHS-ester of fluorescein, available fromMolecular Probes, Eugene, Oreg.) which is attached to the 5′ end of thetag complement through a polyethylene glycol linker (ClonetechLaboratories, Palo Alto, Calif.). The sequences of the tag complementsare simply the 12-mer complements of the tags listed above.

A flow chamber of the design shown in FIGS. 2A and 2B is employed inassociation with an Olympus Optical Co., Ltd. (Tokyo, Japan) modelBX60MF5 fluorescent microscope fitted with a model U-ULS75XE 75 wattXenon arc lamp, a motorized filter wheel, a Ludl Electronic Products,Ltd. computer-controlled stage, and a Photometries, Ltd. (Tucson, Ariz.)PXL CCD camera with a 2000×2000 pixel array. Appropriate bandpassfilters (122) and (124) are employed for exciting fluorescein andtransmitting fluorescent signal to CCD camera (120). Microparticiepositions are determined by top-lighting with broadband light from Xenonlamp (126) reduced by a factor of about 10⁻⁴ with a neutral densityfilter. Fluorescent images are collected with about 2 minute exposuretimes.

Height (204) of flow chamber (201) is selected to be 7 μm, orapproximately 140% of the diameter of the GMA beads. Width (210) of flowchamber (201) is selected so as to ensure that a 3×3 array of 9 imagepixels will cover approximately 40-60% of a bead's image after 10×magnification (as illustrated in FIG. 7). Thus, in order to captureimages of tiles of about 100 thousand 5 μm GMA beads, width (210) isselected to have a value of 1.7 mm. Length (212) is selected so that theflow chamber can hold from 1 to 10 tiles of about one hundred thousand 5μm diameter beads each. The cross section (220) of inlet passage (214)matches that of the inlet tubing and gradually enlarges to match that offlow chamber (201) in the region of the planar cavity, i.e. the regionholding the GMA beads on which analysis is performed. It is desirable tohave a constant cross section through the planar cavity of flow chamber(201) to minimize the creation of non-uniform flow patterns, as mightoccur with sudden constrictions and/or expansions in cross section. Bothbody (218) and cover (216) of flow chamber (201) are glass, and theplanar cavity and channels of body (218) are formed by standard chemicaletching techniques. Cross section (222) of outlet passage (224) isselected to match the cross section of flow chamber (201) at dam (202).

The fluidics system of FIG. 5 a which includes all valves, syringe pump(500), and Peltier block (152), is controlled by code written in LabVIEW5.0 (National Instruments, Austin, Tex.) and run on a Compact DeskproPentium-based microprocessor, which is connected to the variouscomponents of the fluidics system by standard 110 circuit boards.Detection system (114) and overall control of the instrument is effectedthrough a Sun Microsystems (Mountain View, Calif.) Sparcstation 5.

Three cycles of ligation, identification, and cleavage are carried outin flow chamber (201) to give the sequences of 12 nucleotides at thetermini of each of approximately 500,000 cDNAs. That is, five tiles ofGMA beads are analyzed in the following series of process steps:

-   -   1. Calibrate focal plane of GMA beads.    -   2. Hybridize decoder.    -   3. Autofocus on tile 1.    -   4. Set focus to bead centers.    -   5. Collect fluorescent image.    -   6. Set focus to bead focal plane (scatter centers).    -   7. Collect image.    -   8. Repeat steps 4-7 for remaining tiles.    -   9. Wash.    -   10. Repeat steps 2-9 for remaining decoders.    -   11. Cleave encoded adaptor.    -   12. Wash.    -   13. Ligate top strand of next encoded adaptor.    -   14. Wash.    -   15. Repeat steps 13-14.    -   16. Kinase bottom strand of encoded adaptor.    -   17. Wash.    -   18. Ligate bottom strand of encoded adaptor.    -   19. Wash.    -   20. Repeat steps 2-9.    -   21. Repeat steps 11-19 for next encoded adaptor.

In steps 2-9, nucleotides of the cDNAs are identified by hybridizing tagcomplements to the encoded adaptors. Specifically hybridized tagcomplements are detected by exciting their fluorescent labels withillumination beam (110) from Xenon arc lamp (126). In step 13, encodedadaptors and T4 DNA ligase (Promega, Madison, Wn at about 0.75 units perμL are passed through the flow chamber at a flow rate of about 1-2 μLper minute for about 20-30 minutes at 16° C., after which wash of step14 is executed by flowing, in succession, a solution of Pronase™(Boehringer Mannheim, Indianapolis, Ind.), a salt wash solution, and anethanol wash solution through the flow chamber, all with the same flowrate of 1-2 μL per minute and for durations of 15, 10, and 10 minutes,respectively. The salt wash solution is 150 mM NaCl and 10 mM Tris-HCI(pH 8.5), and the ethanol wash solution is 3:1 (v/v) solution of thesalt wash solution and ethanol. The ligation and wash steps 13 and 14are repeated once, after which the adaptors and the cDNAs are preparedfor second strand ligation by passing T4 DNA kinase (New EnglandBioscience, Beverly, Mass.) at 7 units per μL through the flow chamberat 37° C. with a flow rate of 1-2 μL per minute for 15-20 minutes.Ligation of the second strand is carried out by flowing T4 DNA ligase(0.75 units per mL, Promega) through the flow chamber for 20-30 minutesat a rate of 1-2 μL per minute, followed by Pronase™ treatment andwashing as described above. Tag complements at 25 nM concentration arepassed through the flow chamber at a flow rate of 1-2 μL per minute for10 minutes at 20° C., after which the fluorescent labels carried by thetag complements are illuminated and fluorescence is collected. The tagcomplements are melted from the encoded adaptors by passing NEB #2restriction buffer with 3 mM MgCl₂ through the flow chamber at a flowrate of 1-2 μL per minute at 55° C. for 10 minutes. Encoded adaptors arecleaved from the cDNAs by passing Bbv I (New England Biosciences,Beverly, Mass.) at 1 unit μL at a flow rate of 1-2 μL per minute for 20minutes at 37° C., followed by Pronase™ treatment and washing, asdescribed above.

What is claimed is:
 1. A method of sequencing multiple targetpolynucleotides in parallel using a sequencing system, comprising:providing a flow chamber comprising a plurality of ridges within theflow chamber, the flow chamber comprising multiple microparticlesimmobilized between the ridges, each microparticle of the multiplemicroparticles including a target polynucleotide to be sequencedimmobilized on the microparticle, the average distance between centersof adjacent microparticles being less than two microparticle diameters,and the ridges being of a height less than a diameter of a microparticleand of a length greater than a plurality of said diameters; performingsuccessive cycles of a base-by-base sequencing methodology for thetarget polynucleotides immobilized on the microparticles in the flowcell; and obtaining data that provides information about the pluralityof target polynucleotides based on the successive cycles of abase-by-base sequencing methodology.
 2. The method of claim 1, whereinthe plurality of ridges within the flow chamber run parallel to the axisof the flow chamber.
 3. The method of claim 1, wherein the successivecycles of a base-by-base sequencing methodology comprise: providing anoligonucleotide through an inlet of the flow chamber; ligating theoligonucleotide to a strand hybridized to a polynucleotide of theplurality of target polynucleotides when the oligonucleotide iscomplementary to a single-stranded portion of the polynucleotide; andcleaving at least a portion of the oligonucleotide that was ligated. 4.The method of claim 3, further comprising: providing a secondoligonucleotide through the inlet of a flow chamber after the cleavingof the at least a portion of the oligonucleotide that was ligated;ligating the second oligonucleotide to the strand hybridized to apolynucleotide of the plurality of target polynucleotides when theoligonucleotide is complementary to a second single-stranded portion ofthe polynucleotide; and obtaining data that provides information aboutthe plurality of target polynucleotides based on the ligation of thesecond oligonucleotide.
 5. The method of claim 1, wherein said obtainingdata comprises using a data processor configured to receive data fromthe successive cycles of the base-by-base sequencing methodology toprovide sequence data for the polynucleotides immobilized on themultiple microparticles.
 6. The method of claim 5, wherein the dataprocessor is further configured to track each microparticle throughmultiple rounds of label identification.
 7. The method of claim 5,wherein the data processor is configured to receive data correspondingto the polynucleotides as they are processed in parallel.
 8. The methodof claim 1, wherein the number of microparticles per unit area in theplanar array is at least eighty percent of the number of microparticlesin a hexagonal array of equal area.
 9. The method of claim 1, whereinthe ridges are spaced such that the microparticles are arrangedsingle-file between the ridges.
 10. The method of claim 1, whereinmultiple ridges in the flow chamber are parallel to each other.
 11. Themethod of claim 1, wherein a distance between ridges is configured toallow for maximal packing of microparticles.
 12. The method of claim 1,wherein a diameter of each of the multiple microparticles isapproximately a same diameter.
 13. The method of claim 1, wherein themultiple microparticles are chemically constrained in the flow chamber.14. The method of claim 13, wherein the multiple microparticles areconstrained by a crosslinked chemical attachment.
 15. The method ofclaim 1, wherein said obtaining data comprises using a light sourceconfigured to provide an excitation beam and an optics system configuredto receive light produced in response to the excitation beam.
 16. Themethod of claim 1, wherein the sequencing system comprises a bodyattached to a plate to form the flow chamber.
 17. The method of claim 1,wherein the multiple microparticles are connected to the flow chamber ina monolayer between the ridges, said monolayer comprising multiple tilesand a detector is configured to separately generate data based on afirst subset of the multiple microparticles and a second subset of themultiple microparticles physically separate from the first subset of themultiple microparticles.
 18. The method of claim 1, wherein at least500,000 microparticles are immobilized in the flow chamber, each of the500,000 microparticles comprising an analyte to be sequenced immobilizedon the microparticle.
 19. The method of claim 1, wherein the distancebetween the elongated ridges having multiple microparticles immobilizedtherebetween is greater than the diameter of a microparticle.
 20. Themethod of claim 1, wherein the microparticles comprise a hexagonalarray.